Reverse transcriptase is an integral part of many oncogenic viruses, including those from human leukemia and AIDS virus, which is essential for viral replication. Long range goals of this proposal are to understand and define the biochemical, enzymological and structural properties of reverse transcriptase so that molecular mechanisms underlying reverse transcription could be clarified. The major objective, therefore, of this research is to identify and define structural domains of reverse transcriptase which are involved in the binding of substrate dNTPs and template- primer as well as those responsible for the expression of reverse- transcriptase associated Ribonuclease H. The results are expected to clarify the basic mechanisms of enzymatic synthesis of DNA catalyzed by reverse transcriptase and may provide an insight into unique properties/structures which may help in designing the potential therapeutic agents. We have analyzed mechanism of anti-reverse transcriptase activity of a number of inhibitors and have identified some reagents which appear to react with reverse transcriptase in a site-specific manner. In addition, we have standardized protocols for covalently linking substrates, template-primers and specific reagents to the enzyme protein. These reagents are quite useful in establishment of site-specific covalent modification of RT which in turn will permit labeling, identification and isolation of peptides carrying these sites. In this manner, we hope to identify domains responsible for the polymerase and RNAse H activity of MuLV and HIV reverse transcriptase. This knowledge will be very useful in understanding of precise mechanisms of many anti-viral (including anti-AIDS) drugs with presumptive anti-RT activity.